Abstract Abnormal oncogenic activation of epidermal growth factor receptor (EGFR) signaling is a key trigger in inducing various types of cancers including lung cancer, which is recognized as one of the most important active targets for NSCLC treatment. The application of EGFR-targeted inhibitors has provided significant benefit for lung cancer patient treatment. However, the acquisition of resistance to EGFR-targeted inhibitors in cancer cells is the major challenge for clinicians and oncologists. In addition to known genetic alterations such as EGFR secondary mutations causing EGFR-TKI resistance, compensatory activation of signaling pathways without interruption of genome integrity remains to be defined. Our preliminary data showed a persistent S6K1 activation in resistant non-small lung cancer cells upon EGFR-TKIs treatment, but not in sensitive cells. Ribosomal protein S6 kinase 1 (S6K1, RPS6KB1), a member of AGC family of serine/threonine protein kinases and a key effector of the mTORC1 (mammalian target of rapamycin complex 1), is known to regulates protein synthesis, cell cycle, cell growth and survival in response to growth factors, and cytokines under both physiological condition and pathological condition. Deregulation of S6K1 is associated with metastasis and poor prognosis in lung, colorectal, ovarian, and breast cancer. However the link of S6K1 with drug resistance remains to be elucidated. We hypothesize that S6K1 activation contributes to the acquired resistance to EGFR target therapy in NSCLCs. We plan to test this hypothesis through three aims. Aim 1 is to investigate if S6K1 activation is a new mechanism of acquired resistance to EGFR target therapy in NSCLC. Aim 2 is to investigate molecular mechanism of EGFR-TKI- resistance through S6K1 pathway. Aim 3 is to investigate the effect of S6K1 inhibitor PF4708671 for overcoming EGRF-TKI resistance. The proposal will help to understand new mechanism in response to EGFR-TKI and to identify S6K1 and its signaling as potential new druggable target(s). The successful completion of proposed study will provide a potential therapeutic strategy by overcoming EGFR-TKI resistance for lung cancer treatment in the future.